We have developed a selection system for producing modifications of yeast alcohol dehydrogenase that affect turnover number, binding constants and subunit cooperativity. Allyl alcohol selection on petite cells results in a high proportion of structural gene mutations at the ADH-I locus affecting these properties. The source of the resistance to allyl alcohol is an enzyme-mediated change in the redox balance of the cell. Two of the amino acid substitutions responsible for this resistance have been identified. One of the exchanges is to an amino acid which is found at the corresponding position in the mammalian enzyme. Many mutants of this enzyme have been isolated, and other classes of conditional and unconditional mutants can be produced. Identification of the altered residues will be carried out in collaboration with Dr. Hans Jornvall. Pure isozyme I is being produced using genetically defined strains, and will be used in a large-scale attempt at crystallization and determination of the tertiary structure by Prof. C.-I. Branden. Other modes of selection, particularly through the use of inhibitors such as pyrazole, will be used to investigate the function of these molecules. Since the redox balance and the NADH binding are governing factors in mammalian cells in determining the rapidity with which the corresponding mammaian enzyme functions as a detoxifying agent in alcohol poisoning, our ability to change these factors in this model system is likely to lead to findings of clinical significance. A greater understanding of the mechanisms of enzyme inhibition and substrate specificity will enable more efficient and specific inhibitors of the reaction to be designed.